This invention relates to methods for the prophylactic and therapeutic treatment of atherosclerosis and to diseases relating to a deficiency in lecithin-cholesterol acyltransferase activity.
Atherosclerosis is a pathological condition of mammals characterized by the accumulation of cholesterol in the arteries. Cholesterol accumulates in the foam cells of the arterial wall, thereby narrowing the lumen. This results in decreased flow of blood. The clinical sequelae of atherosclerosis include heart disease and heart attack, stroke, and peripheral vascular disease. Together, these diseases account for more disease-related deaths in industrialized countries than any other cause.
The development of human atherosclerosis is inversely related to the concentration of high density lipoproteins (HDL) in the serum. D. J. Gordon and B. M. Rifkind (1989) N. Engi. J. Med. 321:1311. High concentrations of HDL appear to protect against the development of premature atherosclerosis, while low HDL cholesterol concentrations are associated with an increased risk of cardiovascular disease. D. J. Gordon et al. (1986) Circulation 74:1217. It has been proposed that a 1% increase in the concentration of HDL would lead to a 3% reduction in risk for developing clinical atherosclerosis in man. Gordon and Rifkind, supra.
The plasma protein enzyme lecithin-cholesterol acyltransferase (LCAT) catalyzes the transfer of fatty acid from the sn-2 position of lecithin to the free hydroxyl group of cholesterol. J. A. Glomset et al. (1966) J. Lipid Res. 7:638). J. McLean et al. (1986) Proc. Nat""l. Acad. Sci. USA 83:2335-2339 described the cloning and sequence of a human LCAT cDNA. J. McLean et al. (1986) Nucl. Acids Res. 14:9397-9406 described a complete gene sequence for human LCAT.
It was first proposed nearly 30 years ago that the esterification process with this enzyme could be the key step in transferring cholesterol from the tissues of the body to the liver. This process, termed xe2x80x9creverse cholesterol transportxe2x80x9d (J. A. Glomset (1968) J. Lipid Res. 9:155), was proposed to facilitate the removal of cholesterol from the body. However, increases in LCAT were not known to diminish the risk of atherosclerosis.
Various mutations of the LCAT gene are known. Individuals who are homozygous for a non-functional LCAT mutant have classic LCAT deficiency disease, characterized by clouding of the cornea, normochromic anemia and glomeruloscierosis. Mutations in the LCAT gene that result in some residual LCAT activity lead to Fish Eye disease, characterized by opacity of the cornea and hypoalphalipoproteinemia. H.-G. Klein et al. (1992) J. Clin. Invest. 89:499-506.
Thus, there is a need for compositions and methods for the prophylactic and therapeutic treatment of atherosclerosis and conditions associated with LCAT deficiency. This invention satisfies this need by providing compositions and methods for increasing the serum level of LCAT activity.
It has been discovered that increasing the level of lecithin-cholesterol acyltransferase activity in a rabbit (which is an accepted model of the development of atherosclerosis in humans; D. J. Gordon and B. M. Rifkind (1989) N. Engi. J. Med. 321:1311), causes a decrease in the accumulation of cholesterol in the arteries. This discovery is surprising because no prior results had indicated that increasing the level of LCAT activity would have such an effect. In the rabbit model, increasing the mass quantity of human LCAT in the serum by about five times above the normal human level also led to significant decreases in total triglycerides, at least five-fold increases in the amount of high density lipoproteins and about a seven-fold decrease in the ratio of total cholesterol to high density lipoproteins in animals fed a high cholesterol diet. Therefore, increasing LCAT activity in the serum of humans, rabbits and other mammals with similar modes of lipoprotein metabolism is an effective treatment against atherosclerosis.
This invention provides methods for treating atherosclerosis in a mammalian subject, including humans, comprising the step of increasing the LCAT activity in the serum of the subject to a level effective to decrease the accumulation of cholesterol in the subject, whereby decreasing the accumulation of cholesterol provides a treatment for atherosclerosis.
In one aspect, the method involves administering to the subject a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a pharmacologically effective amount of LCAT. In one embodiment, the composition is administered intravenously. This invention also provides pharmaceutical dosage forms comprising a pharmaceutically acceptable carrier and a pharmacologically effective amount of LCAT.
In another aspect, the method involves transfecting cells with a nucleic acid comprising a nucleotide sequence coding for expression of LCAT, whereby the transfected cells express LCAT and secrete sufficient LCAT into the serum to increase LCAT to a level effective to decrease the accumulation of cholesterol. In one embodiment, the method involves transfecting cells in vivo. In another embodiment, the method involves transfecting cells ex vivo and administering transfected cells that express and secrete LCAT to the subject in an amount sufficient to increase LCAT activity to a level effective to decrease the accumulation of cholesterol.
In another aspect, the methods involve administering a drug that up-regulates the endogenous production of LCAT in the subject.
In another aspect, the method involves increasing both the serum LCAT activity and the level of Apo A-I in the serum to an amount effective to decrease accumulation of cholesterol. The invention also provides vectors comprising a nucleic acid that comprises expression control sequences operatively linked to a sequence that codes for the expression of LCAT and expression control sequences operatively linked to a sequence that codes for the expression of an Apo A-I.
This invention also provides methods for treating an LCAT deficiency condition in a mammalian subject comprising increasing the LCAT activity in the serum of the subject to a therapeutically effective level. The condition can be Fish Eye Syndrome or Classic LCAT Deficiency Syndrome.
This invention also provides non-human mammals transgenic for LCAT having an absolute serum LCAT activity of at least 1000 nmol/ml/hr.
FIG. 1 depicts a comparison of the atherosclerosis in control and transgenic rabbits over-expressing human LCAT by quantitative planimetry. The aortas of LCAT transgenic and control male rabbits, fed a 0.3% cholesterol-chow diet for 17 weeks, were harvested and stained with Sudan IV. The percent of the surface area that stained was determined by planimetry of the digitized image. J. F. Cornhill et al., (1985) Arteriosclerosis, 5:415. Top panel: Planimetry of aortas of transgenic rabbits (n=12). Middle panel: Planimetry of aortas of control rabbits (n=10). The compilations of the images from the study groups are summarized for transgenic and control rabbits. Bottom panel: Graded shades of the probability of distribution.
FIG. 2. Photographs of cross sections of aorta from control and transgenic rabbits fed a high cholesterol diet. A 1 mm section was taken at the descending thoracic aorta at the same position for each aorta, stained with PAS, and the degree of foam cell accumulation in the intima of the controls was compared to the lack of intimal cell formation or change in intimal cell thickness in the transgenic aortae. Left hand photograph: Cross section of aorta of control rabbit. Right hand photograph: Cross section of aorta from transgenic rabbit.
FIG. 3 shows the extent of intimal cellular proliferation as shown in FIG. 2, quantitated using a ratio of the intima to media. A. V. Chobanian et al., (1989) Hypertension 14:203. Left hand graph: Quantitative assessment of the intimal/media ratio (p less than 0.003). Right hand graph: Quantitative assessment of the percent of surface area (p less than 0.009). In both cases, the quantitative assessment was significantly lower in the transgenic LCAT rabbits than in controls.
FIG. 4 shows the significant ( less than 0.05) bivariate Pearson correlations of post-diet variables with aortic atherosclerosis in control and transgenic rabbits. The intima/media ratio correlated well with the planimetry assessment of atherosclerosis in both the control group and the entire study. FIG. 4A: Control group, showing % surface area, FIG. 4B: control group, showing LCAT activity in nmol/ml/h, FIG. 4C: control group, showing non-HDL cholesterol, in mg/dl; FIG. 4D: control group, showing ratio of total cholesterol to HDL cholesterol. FIG. 4E: entire study group (controls and transgenics), showing % surface area, FIG. 4F: entire group, showing LCAT activity in nmol/mlh, FIG. 4G: entire group, showing non-HDL cholesterol, in mg/dl; FIG. 4H: entire group, showing ratio of total cholesterol to HDL cholesterol. The intima/media ratio was inversely correlated with the severity of atherosclerosis (see FIGS. 4B and 4F) and positively correlated with the non-HDL cholesterol (see FIGS. 4C and 4G) and total cholesterol/HDL cholesterol (TC/HDL) (see FIGS. 4D and 4H).
FIG. 5 depicts the nucleotide sequence and deduced amino acid sequence of a genomic clone encoding a human LCAT (SEQ ID NO:1). FIG. 5A: Nucleotide sequence of nucleotides 1-1092, deduced amino acid sequence and exon start and start site. FIG. 5B: Nucleotide sequence of nucleotides 1093-2100, deduced amino acid sequence, and exon start and stop sites. FIG. 5C: Nucleotide sequence of nucleotides 2101-3108, deduced amino acid sequence, and exon start and stop sites. FIG. 5D: Nucleotide sequence of nucleotides 3109-4368, deduced amino acid sequence, and exon start and stop sites. FIG. 5E: Nucleotide sequence of nucleotides 4368-5292, deduced amino acid sequence, and exon start and stop sites. FIG. 5F: Nucleotide sequence of nucleotides 5293-6552, deduced amino acid sequence, and exon start and stop sites. FIG. 5G: Nucleotide sequence of nucleotides 6553-6901 FIG. 5F: Nucleotide sequence of nucleotides 5293-6552, deduced amino acid sequence, and exon start and stop sites.